Fig 2: LPD impairs glycolytic and carbohydrate metabolism in undifferentiated LPD ESC lines. A-C Metabolomics analysis of mean concentrations (protein normalised) of key metabolites involved in (A) glycolysis, (B) fructose, mannose, pentose pathways, and (C) TCA pathways from lysates of undifferentiated male NPD and LPD mESCs (PN 10–12; n = 7) cultured in standard mESC medium (KnockOut DMEM + LIF + KO/SR). * P < 0.05, ▽ P < 0.1. G 6-P = glucose 6-phosphate; F 6-P = fructose 6-phosphate; F 1,6 bP = fructose 1,6-bisphosphate; DHAP = dihydroxyacetone phosphate; 3-PGlyc = 3-phosphoglycerate; PEP = phosphoenolpyruvate; M 6-P = mannose 6-phosphate; G.amine 6-P = glucosamine 6-phosphate; Glyc. = glycerate; 6-P Gluc = 6-phosphogluconate; Ribo 5-P = ribose 5-phosphate; Sedo 7-P = sedoheptulose-7-phosphate; -ketoglut = alpha-ketoglutarate. D, E RNAseq above and qRT-PCR analysis below of gene expression of glycolytic enzymes (D) and other carbohydrate metabolism regulators (E) in undifferentiated LPD and NPD ESC lines (PN 9–12; n = 5) cultured in standard mESC medium (KnockOut DMEM + LIF + KO/SR). qPCR gene expression is normalised with 3 house keeping genes Sdha, Tbp and Tuba-1. * = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001, ▽ P < 0.1. Hk1, Hk2, Hk4 = hexokinase 1,2 or 4; Gpi = glucose phosphate isomerase; PfkP, PfkM, PfkL = phosphofructokinase P, M or L variants; Aldoc = aldolase C; Pkm = pyruvate kinase M; Hkdc1 = hexokinase domain containing 1; Khk = ketohexokinase; Mpi = mannose phosphate isomerase; Pfk1-4 = phosphofructokinase 1–4 variants; Pgls = phosphogluconolactonase; Rpe = ribulose-phosphate 3-epimerase; Glut5 (Slc2a5) = glucose transporter 5; Aldh2 = aldehyde dehydrogenase 2; Prprsap1 = phosphoribosyl pyrophosphate synthetase-associated protein 1; Fbp2 = fructose-bisphosphatase 2; Gale = UDP-galactose-4-epimerase; Pgm2 = phosphoglucomutase 2; Gaa = acid alpha-glucosidase. F Glycolytic enzyme activity assays on hexokinase (HK) and phosphofructokinase (PFK) from LPD and NPD ESC lines. Data, normalised by protein (BCA Protein Assay), presented as means ± SEMs based on n = 6 for PFK and n = 7 for HK analysis. *P = 0.029

Fig 3: Mutant IDH1 activates glycolysis in IBOs through the upregulation of Pfkp. (A) The level of Pfkp expression in the indicated IBOs by RT-qPCR (n = 4, *P < 0.05, NS not significant). (B) Protein expression levels of Pfkp, Pfkm, and Pfkl as determined by western blotting. (C,D) Enzymatic assay of PFK-1 activity in the indicated IBOs. Time course of optical absorbance at 450 nm in (C), and comparison of PFK-1 activity in (D) (n = 4, *P < 0.05, **P < 0.01). (E) Gene expression of Pfkp in mut-IBOs transfected with shRNA for Pfkp (shPfkp-2, 5) or scrambled shRNA (Scr) as control (n = 4, *P < 0.05). (F) Organoid-forming efficiency of the indicated IBOs (>100 μm) at 7 days after plating (n = 4, 3000 cells per group, *P < 0.05). (G) Gene expression of Pfkp in mut-IBOs treated with 20 μM AGI-5198 (+) or DMSO vehicle (−) (n = 4, *P < 0.05). (H) IBOs established from wild-type mice were treated with 10 mM 2-HG (+) or vehicle (−) upon serial passage. Gene expression of Pfkp at passage 4 by RT-qPCR (n = 3, P = 0.14). (I) The level of trimethylation of histone H3 lysine4 (H3K4me3) on the promoter region of Pfkp by ChIP analysis in IBOs. The upper panel shows the schematic diagram of the transcriptional start site (TSS) and amplicons (0.2 kb and 50 kb downstream of the TSS). The lower graph shows ChIP- qPCR data (relative to input DNA, n = 4, *P < 0.05).

Fig 4: Inhibition of GAPDH induces changes similar to those caused by chronic hyperglycaemia.a Schematic of glycolysis showing where Koningic acid acts. a, b Glycolytic (a) and TCA cycle (b) metabolite abundances in LG-cells cultured for 48 h without (black) or with (blue) 5 µM koningic acid (KA) and subsequently stimulated with 2 mM or 20 mM glucose in the absence of KA (n = 3 biologically independent experiments). c, d Insulin secretion (c) and insulin content (d) in HG-cells (red) and in LG-cells cultured in the absence (black) or presence (blue) of 5 µM KA for 48 h and then stimulated with 2 mM or 20 mM glucose in the absence of KA (n = 3 biologically independent experiments). e, f mRNA levels for the indicated glycolytic (e) and mitochondrial (f) genes assessed by qPCR in LG-cells cultured in the absence (black) or presence (blue) of 5 µM KA. Pfkl, Mdh2 and Ndufs8, n = 3 biologically independent experiments; Eno1, n = 4 biologically independent experiments; Pdk1, n = 5 biologically independent experiments; Pfkfb3, Pfkl, Aldob, Idh2, Sdha and Ndufa4, n = 6 biologically independent experiments. All panels show individual data points and mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed unpaired Student’s t test. Source data are provided as a Source Data file.